Get Your Dual Luciferase Assay On
'Dual Luciferase Assay Protocol '(Promega E1910) V2.0 * Make overnights of your samples: ** Make serial dilutions of WT or YFG strains that have been transformed with either the pVW31 or pVW32 plasmids. These plasmids have a URA marker so the cells should be grown in C –URA Media (be sure the C –URA has 2% glucose or another carbon source so the yeast will grow). Grow the samples overnight in the 30 degree shaking incubator. ** Be sure to include the appropriate amount of samples. There should be one set of serial dilutions for every strain and/or for every drug concentration to be tested as well as control samples. The controls will include a negative control (WT+pVW31 if you are testing YFG strains or samples without drugs) and the positive control WT+pVW31 that will be treated with 8-aza-a. * In the morning, measure the OD600 of your samples using the Nanodrop. Be sure that you press the “use cuvette” when measuring your sample. Be sure to blank the Nanodrop with whatever you overnighted your cells in. If you need some help with the Nanodrop, read this first. Be sure to record the OD600 for all of you samples. You will want to dilute your cells to an OD6000=0.2/mL in 4mL of MinD+. This can be done easily in an Excel sheet or by hand. WHEN YOU SPIN DOWN YOUR SAMPLES, THEY ALL NEED TO HAVE THE SAME VOLUME, USE WATER OR C-URA TO MAKE UP FOR ANY DIFFERENCES ** EXAMPLE CALCULATION: You get a Nanodrop OD600 aka A600 reading of 0.4. This is 0.4/ml since you read 1ml. You need 0.2/ml * 4ml, or 0.8 OD600 worth of yeast, so spin down 2mls of your overnight (2mls * your overnight OD of 0.4/ml = 0.8), then re-suspend this pellet in 4ml of MinD+ to have 0.2/ml yeast in your MinD+. We have a spreadsheet with formulas typed in for this as well. REMEMBER: this example calculation works if your yeast right out of the tube is in the 0.1-1 reange for OD600. If not, you will have to dilute the yeast, get diluted OD600, calculate undiluted OD60, and go from there, as detailed under Measuring OD600 on the Nanodrop ** Suggestion: Prepare cell culture tubes with 3ml MinD+. When you calculate how much of each overnight you need, transfer that volume to a microcentrifuge tube and spin it down at max speed for 2 minutes. When the samples are done spinning, remove all of the remaining C-URA with a p1000. Re-suspend the cells in the remaining 1mL or MinD+ (or MinD+-volume of drug) and then transfer the re-suspended cells to the culture tube with 3mL MinD+. REMEMBER YOUR STERILE TECHNIQUE AND MAKE SURE YOU DON’T MIX UP SAMPLES * This is the point in the protocol where you will add any drugs you may be using. To the MinD+, add the appropriate amount of 8-aza-a for your positive control. Be sure to have your drug dilutions prepared ahead of time. Also, be sure to not re-suspend the yeast in the drug solution! This may kill them all and you will waste sooooo much time. Instead, add the drug to the MinD+ after the yeast have been re-suspended. * Incubate the cells in the 30 degree incubator for 4 hours. However, you will want to set a 3 hour timer. * After 3 hours you will want to start a 1 hour timer and then you are going to want to prepare your buffers for the rest of the protocol. Go to the -80C freezer and get out either a fresh, or pre-mixed set of buffers (Luciferase Assay Solution and Stop n’ Glo) and PLB (Passive Lysis Buffer). SOMETIMES THESE CAN BE FOUND IN THE -20C FREEZER! ** If you are using pre-mixed buffers, great! Just sit them on ice to they can thaw out slowly. You will know if they have been mixed because they will be opened, have a date written on them and are a funny green color. ** If you are mixing fresh buffer, that’s also great! There are 4 bottles/tubes you will need to grab, and lucky for you, they are color coordinated. *** LUCIFERASE ASSAY SOLUTION: These are the green bottles. Re-suspend all of the Luciferase Assay Substrate in the full 10 ml of Luciferase Assay Buffer by adding 1ml of Luciferase Assay Buffer to the brown cap with the tablet, dissolve the tablet and move the Buffer back into plastic vial. Repeat a few times to be sure that you dissolved all of the tablet. Store in the screw-cap white plastic vial the Luciferase Assay Buffer came in *** STOP N’ GLO: These are in the blue bottles. Re-suspend all of the Stop n' Glo substrate in the Stop n' Glo buffer, and store in the screw-cap white plastic vial the Stop n' Glo buffer came in ** For the PLB, you will want to make a 1X stock from the 5x stock in the freezer. For example, if you are going to need 5ml of stock, you can mix 1ml of PLB with 4ml dH2O. You will be using 500ul per sample, so keep that in mind when diluting you stock. Keep the 1x PLB on Ice. * When the 1 hour timer has gone off, go get your cells from the incubator. You are going to have to get the OD600 for all of your samples all over again. This time, you are going to want to spin down a total OD600=0.22/ml at maximum speed for 2 minutes. WHEN YOU SPIN DOWN YOUR SAMPLES, THEY ALL NEED TO HAVE THE SAME VOLUME, USE WATER OR C-URA TO MAKE UP FOR ANY DIFFERENCES ** SAMPLE CALCULATION: You check your MinD+ culture after shaking in the Nanodrop, and get 0.3 in 1ml cuvette, or 0.3/ml. REALITY CHECK: you just put in 0.2/ml 4 hours ago, so if you read <0.2ml after 4 hours of shaking you’re doing something wrong! Okay. So you have 0.3/ml- you want a total of 0.22, so you spin down 0.22*1ml/0.33 = 2/3 ml or 667ul to get the pellet. * Carefully remove all of the medium from the spin downs and re-suspend the cells in 500ul of your freshly prepared 1x PLB. * Lyse the cells for 30 minutes at room temperature. * While you are waiting, make the template for the plates. See the luminometer template for an idea of the layout. ** Something to think about: the Renilla levels are much higher and, because of light scattering, can interfere with the much lower firefly reading of the next sample. Therefore, you need to space the samples out in the 96-well format. ** REMEMBER TO HAVE PLB ONLY WELLS!!! SEE MARK OR CHRISTINE FOR DIRECTIONS ON HOW TO RUN THE ASSAY ON VICTOR AND HOW TO CLEAN THE MACHINE WHEN YOU ARE DONE!!!! Part Numbers! Black 96-well plates (Nunc 267342 U96 PP 0.5ml) Promega Dual-Luciferase Assay Kit (Product Number E1910, E1960 AND E 1980)